The objective of Project 2 is to characterize the involvement of MBT (malignant brain tumor domain) proteins in prefrontal cortex (PFC) and nucleus accumbens (NAc) in mediating depression- and antidepressant-like responses in animal models. MBT proteins are the best known readers-i.e., effectors-for several key methylation states of histones, including repessive histone methylation at Lys 9 of histone H3 (H3K9me2). However, virtually nothing is known about the function of MBT proteins in brain. We have found that 3 MBT proteins, L3MBTL1, L3MBTL2, and SFMBT1, are highly expressed in PFC and NAc, where they display dramatic regulation in response to several forms of chronic stress. Depressed humans show similar altered levels of some of these same MBT proteins. Moreover, mice lacking L3MBTL1 show a pro-depression-like phenotype, consistent with findings in Project 1 that downregulation of H3K9me2 increases susceptibility to chronic stress. We have generated mutant lines of the other MBT proteins and now propose the comprehensive characterization of: 1) the regulation of L3MBTL1, L3MBTL2, and SFMBT1 in our Center's battery of depression models, and 2) the behavioral phenotypes of conditional and brain region-specific knockout, or overexpression, of these three MBT proteins. We will then use our novel method, which permits the genome-wide analysis of chromatin modifications specifically within adult PFC neurons, to map the binding of MBT proteins and their key target sites of histone methylation, including H3K9me2, in PFC neurons in chronic stress models, with parallel studies performed on PFC neurons from depressed humans (Project 4). We will also study conditional knockouts of several key histone methyltransferases, including G9a (with Project 1), which catalyze the methylated histone sites read by MBT proteins, based on the hypothesis that similar phenotypes will be observed. We are particularly excited about comparing preclinical and clinical chromatin datasets through which we will construct, specifically for PFC neurons of mouse and human, a genome-wide map of epigenetic risk loci highly relevant for depression. Together, this work provides a template for the analysis of the role played by other histone reader proteins in depression. RELEVANCE (See instructions): Depression has a lifetime risk of ~15% for the U.S. general population, yet available antidepressant therapies are based on serendipitous discoveries over 6 decades ago, and fully treat <50% of all affected individuals. An improved understanding of the molecular basis of depression will lead to improved treatments and diagnostic tests-a high priority for the National Institutes of Health.